In this K99 proposal, Dr. Luca will investigate how molecular recognition influences ligand selectivity in the Notch pathway, a signaling system of fundamental importance to cell fate determination and the development of many cancers. Dr. Luca will determine the structural and biophysical determinants that distinguish each class of Notch ligand, and will enhance ligand function using techniques rooted in structural and combinatorial biology. The five year training plan outlined in this proposal begins with a mentored phase under the guidance of Prof. K. Christopher Garcia at Stanford, where Dr. Luca will expand his expertise in protein engineering, biophysics, and structural biology. This experience will establish Dr. Luca as an independent investigator with the experimental range to address difficult problems in ligand engineering and cancer biology. Notch signaling is initiated when a Notch receptor on the surface of a cell engages a ligand on an opposing cell, triggering a series of proteolytic cleavages required for activation. While the overall signaling mechanism is conserved, individual Notch receptors and the two classes of Notch ligands, Jagged and Delta- like, are able to induce distinct cellular responses in both normal and cancer cells. There is a large body of literature describing pleiotropic Notch signaling in development and disease, yet we poorly understand the events that govern ligand and receptor-specific effects. Structural studies of Notch receptor-ligand interactions would clarify this process, but these experiments are difficult because their nearly undetectable affinity precludes reconstitution of stable complexes. Dr. Luca recently overcame this obstacle using directed evolution to affinity-mature Notch1 interactions with Delta-like 4 (DLL4), which stabilized complexes for co-crystallization. The resulting structure provided the first and only visualization of a Notch receptor-ligand interface. During the mentored phase of this proposal, Dr. Luca will build upon this transformative result by investigating how structural and biophysical parameters influence differential ligand activities. This will be achieved by 1) evolving Jagged1 (Jag1) variants that bind Notch1 with enhanced affinity, 2) determining a high-resolution crystal structure of the Notch1-Jag1 interacting domains, 3) imaging full-length Notch1-Jag1 or Notch1-DLL4 extracellular domain complexes by electron microscopy and 4) performing mechanoactivation assays to determine the force requirements for Notch activation by Jag1 vs. DLL4. As Dr. Luca transitions to an independent position, he will perform structure-guided engineering of DLL4 proteins to generate high-affinity Notch3-specific DLL4 variants, and evaluate them as inhibitors of non-small cell lung cancer tumor growth in vivo. However, the generation of additional receptor-specific ligands will allow for deep interrogation of Notch biology for years to come. The successful completion of this work will establish the structural and biophysical basis for Notch activation by each class of ligand, and will instruct the engineering of novel modulators of Notch signaling with expanded capabilities in a variety of biological and therapeutic contexts.